Erythrocytic stages of the
malaria parasite Plasmodium falciparum express four related
papain-family
cysteine proteases, termed falcipains.
Falcipain-2 and
falcipain-3 are food vacuole hemoglobinases, but determination of the specific roles of these and other falcipains has been incomplete. To better characterize
biological roles, we attempted disruption of each
falcipain gene in the same strain (3D7) of P. falciparum. Disruption of falcipain-1,
falcipain-2, and falcipain-2' was achieved. In each case knockouts multiplied at the same rate as wild-type parasites. The morphologies of erythrocytic falcipain-1 and falcipain-2' knockout parasites were indistinguishable from those of wild-type parasites. In contrast, consistent with previous results,
falcipain-2 knockout trophozoites developed swollen,
hemoglobin-filled food vacuoles, indicative of a block in
hemoglobin hydrolysis and were, compared to wild-type parasites, twice as sensitive to
cysteine protease inhibitors and over 1000 times more sensitive to an aspartic
protease inhibitor. The
falcipain-3 gene could not be disrupted, but replacement with a tagged functional copy was readily achieved, strongly suggesting that
falcipain-3 is essential to erythrocytic parasites. Our data suggest key roles for
falcipain-2 and
falcipain-3 in the development of erythrocytic
malaria parasites and a complex interplay between P. falciparum
cysteine and aspartic
proteases.